The long term goal is the development of better, longer hypothermic storage conditions, using novel insect antifreeze proteins, for liver transplants than is currently possible in clinical practice. The use of hypothermia as the principal means to suppress metabolism in a reversible way is the foundation of most of the effective methods for tissue and organ storage. Hypothermic injury results in vascular dysfunction during liver storage, therefore vascular tissue models were employed in our Phase I SBIR proposal. Blood vessel rings studies could be performed with the small quantities of recombinant insect-derived antifreeze proteins available with our existing manufacturing methods. A recently discovered insect-derived antifreeze protein formulation was tested sub-zero at -100C and at +40C, to detect antifreeze protein concentration dependent cytotoxicity effects, and compared with controls without antifreeze proteins in order to increase the duration of hypothermic storage. The test temperature, -100C, was selected on the basis of the greater non-colligative freezing point depression produced by the insect antifreeze peptides that provide almost an order of magnitude greater activity compared with other previously described antifreeze proteins derived from other sources. The results demonstrate that our lead antifreeze protein formulation consisting of equal amounts of Dendroides canadensis types 1, 2, 4 and an activity enhancing thaumatin-like protein resulted in preservation of fresh tissue function control values for 3-6 days at -10:C, while tissues stored at +4:C demonstrated statistically significant changes compared with both fresh controls and the -10:C storage group. Consistent storage of tissues without the antifreeze proteins at -10:C was not possible because ~30% of the samples froze. The insect-derived antifreeze proteins inhibited ice nucleation at -100C permitting mammalian tissue storage at much lower levels of endogenous metabolic activity. These results demonstrate proof of feasibility for sub-zero storage of mammalian tissues. In this Phase II SBIR application scale up of antifreeze protein manufacturing and optimization of longer, better storage procedures for mammalian livers are proposed. The above antifreeze combination tested in Phase I, plus additional insect-derived antifreeze protein formulations selected from prior research studies on antifreeze protein activity by our consultant, Professor John Duman, and controls will be compared in four specific aims employing tissue and liver models of increasing complexity and relevance for human liver transplantation. Each model will be evaluated using a panel of assays and histopathology to determine relative function after storage. The antifreeze protein formulations will be tested at -100C and controls will be tested with a currently employed clinical organ storage solution at +40C. In parallel with these aims scale up of antifreeze protein production to achieve the quantities required for storage of large mammal livers, both porcine and human, will be performed by Professor Duman's laboratory. His group will also validate the batches as they are being produced. These studies should lead to longer hypothermic, sub-zero storage periods for tissues and organs. PUBLIC HEALTH RELEVANCE: Livers destined for transplantation are currently stored using static cold storage in a chemically defined solution at 4:C for periods of up to 6 hours. However, the application of static cold storage has proven to be insufficient to fulfill the organ demand for patients with irreversible liver failure, because marginal livers can not be preserved optimally, for long enough, by static cold storage to permit organ evaluation to occur. These limitations should be overcome by use of a lower, sub-zero hypothermic temperature (-10:C) to further reduce metabolism in combination with insect-derived antifreeze proteins to inhibit ice nucleation. This may result in increased availability of organs for transplant.